Adenovirus-medicated gene transfer to cardiac and vascular smooth muscle

ABSTRACT

The present invention relates to the use of adenovirus-mediated gene transfer to regulate function in cardiac and vascular smooth muscle cells. A recombinant adenovirus comprising a DNA sequence that codes for a gene product is delivered to a cardiac or vascular smooth muscle cell and the cell is maintained until that gene product is expressed. Delivery is direct injection into a muscle cell or infusing a pharmaceutical composition containing an adenovirus virus vector construct intravascularly.

This application is a continuation of U.S. Ser. No. 08/972,831, filedNov. 18, 1997, pending, which is a continuation of U.S. Ser. No.08/417,459, filed Apr. 5, 1995, abandoned, which is a continuation ofU.S. Ser. No. 07/977,496, filed Nov. 18, 1992, abandoned.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process of regulating genetranscription and gene product expression in cardiac and vascular smoothmuscle. More particularly, a process of the present invention relates toadenovirus vector mediated delivery of genes to cardiac muscle and tocoronary vascular smooth muscle.

BACKGROUND OF THE INVENTION

Somatic gene therapy can be defined as the ability to program theexpression of foreign genes in non-germ line (i.e., non-sperm and egg)cells of an animal. Methods of somatic gene therapy can be divided intotwo categories. Ex vivo gene therapy involving the removal of cells froma host organism, transfection a foreign gene into those cells, andreimplantation or transplantation of the transformed or transgenic cellsback into a recipient host. In contrast, in vivo gene therapy involvestransfection of a foreign gene directly into cells of a recipient hostwithout the need for prior removal of those cells from the host.

The utility of somatic gene therapy for human subjects is dependent upona number of factors. First, the transfection method must be efficient.Second, expression of the foreign gene should be localized to specifictarget tissues. Third, a given transfection process should be associatedwith a minimal risk of mutating the host cells and of causing apersistent infection of the host organism.

Several possible strategies to introduce genes into tissues of the bodyhave been employed in the past (Stratford-Perricaudet et al., 1990;Rosenfeld et al., 1992; Wolfe et al., 1992). Procedures to introduceforeign genes into cells include direct transfection (Davis et al.,1986) and retroviral gene transfer (Dichek et al., 1991; Wilson et al.,1988a; Wilson et al., 1988b; Kay et al., 1992). In some cases,genetically altered cells have been reintroduced into animals (Dichek etal., 1991; Roy Chowdhury et al., 1991) where their continued functionhas been monitored for variable periods of time.

Recently, adenovirus-mediated gene transfer has been investigated as ameans of somatic gene therapy into eukaryotic cells and into wholeanimals (van Doren et al., 1984a; van Doren et al., 1984b;Ghosh-Choudhury and Graham, 1987; Stratford-Perricaudet et al., 1990;Rosenfeld et al., 1991; Rosenfeld et al., 1992). A problem withadenovirus mediated gene transfer is the low level of gene productexpression in target cells and a resultant lack of a functional effect.

Although adenovirus-mediated gene transfer has been used to treatornithine transcarbamylase (OTC) deficiency in newborn mice, theexpression of the ornithine transcarbamylase enzyme in the virusinfected mice was typically at or below expression levels in normal micewith the result that the defect was only partially corrected(Stratford-Perricaudet et al., 1990). On the basis of those data, onewould not expect that adenovirus-mediated gene transfer would beapplicable to treatment of a disease requiring an overexpression of agene product.

Adenovirus mediated transfer of the gene for cystic fibrosistransmembrane conductance regulator (CFTR) into the pulmonary epitheliumof cotton rats has been attempted, although it has not been possible toassess the biological activity of the transferred gene because there wasno physiologic effect of gene transfer despite expression of the CFRRprotein in lung airway cells (Rosenfeld et al., 1992). Still further,lung expression of α1-antitrypsin protein was not associated with aphysiologic effect (Rosenfeld et al., 1991). Taken together, those datado not demonstrate that adenovirus can transfer genes into cells anddirect the expression of sufficient protein to achieve a physiologicallyrelevant effect.

Targeting somatic gene therapy to cardiac tissue can be used in thetreatment of a number of inherited and acquired cardiac diseases such asgenetic disorders of myocardial cells. By way of example, injection ofthe normal dystrophin cDNA can be used to correct the defects in cardiaccontractility seen in patients with Duchenne's muscular dystrophy. Byway of further example, the injection of plasmids encoding recombinantangiogenesis factors directly into the left ventricular wall can be usedto stimulate new collateral circulation in areas of chronically ischemicmyocardium. Somatic gene therapy can also be used to directly study themolecular mechanisms regulating cardiac myocyte gene expression bothduring cardiac myogeneses and in a variety of pathophysiologic statessuch as cardiac hypertrophy.

As many as 1.5 million patients per year in the U.S. suffer a myocardialinfarction (MI). Many millions more suffer from syndromes of chronicmyocardial ischemia due to large and small vessel coronaryatherosclerosis. Many of these patients will benefit from the ability tostimulate collateral vessel formation in areas of ischemic myocardium.Adenovirus mediator gene transfer methods provide an alternativeapproach to the current methods of coronary artery bypass andpercutaneous transluminal coronary angioplasty. In particular, manypatients have such revere and diffuse atherosclerosis that they are notcandidates for CABG or PTCA. Thus far, there has been no approach whichhas successfully stimulated collateral vessel formation in areas ofischemic myocardium.

Previous approaches have all utilized in vitro transfection protocolsinto neonatal cardiocytes or transgenic approaches in mice. Such studiesare complicated by the fact that neonatal cardiocytes may not reflectthe in vivo situation and by the fact that neonatal cardiocytes have anextremely limited life span in tissue culture and cannot be incorporatedinto the heart. Moreover, transgenic approaches are lengthy (requiring 6months to 1 year) technically difficult and expensive. The gene transferapproach of the present invention provides a solution to these problemsand provides for the stable expression of recombinant gene products incardiac myocytes in vivo.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a process of regulating the function ofa cardiac or a vascular smooth muscle cell, whether that cell be in vivoor in vitro. Muscle cell function is regulated by altering thetranscription of genes and changes in the production of a genetranscription product such as a polynucleotide or a polypeptide. Thatpolynucleotide or polypeptide interacts with the cardiac or vascularsmooth muscle cell to regulate function of that cell.

In one aspect, therefore, the present invention provides a process ofregulating expression of a gene product in a cardiac or a vascularsmooth muscle cell, which process comprises the steps of:

a) delivering to the muscle cell an adenovirus vector constructcomprising a coding sequence that encodes the gene product, which vectordrives expression of the gene product in the muscle cell; and

b) maintaining the muscle cell under physiological conditions sufficientfor the gene product to be expressed in the muscle cell.

An adenovirus used in a process of the present invention isreplication-defective. Preferably, a replication-defective adenoviruslacks the early gene region E1 or the early gene regions E1 and E3. Apreferred adenovirus is a type 5 adenovirus and a coding sequence ispreferably flanked by adenovirus type 5 sequences.

In a preferred embodiment, a coding sequence is operatively linked to anenhancer-promoter other than an adenovirus enhancer-promoter. Apreferred enhancer-promoter is a CMV promoter, a SV40 early promoter, aRSV promoter or a MCK enhancer. More preferably, an enhancer-promoter isa tissue specific enhancer-promoter. A preferred cardiac muscle specificenhancer-promoter is a cTNC promoter. A preferred vascular smooth musclespecific enhancer-promoter is an endothelin promoter or a smooth muscleα-actin promoter.

A coding sequence is operatively linked to a transcription-terminatingregion. A preferred transcription-terminating region comprises an SV40or protamine gene polyadenylation signal. In another preferredembodiment, a coding sequence comprises a cDNA insert.

In a preferred embodiment, a gene product is a polynucleotide. Apreferred polynucleotide is an antisense molecule. A preferred antisensemolecule is an antisense polynucleotide to c-myb.

In another embodiment, a gene product is a polypeptide. A preferredpolypeptide is a growth factor. A preferred such growth factor is FGF-5,acidic FGF, basic FGF or PDGF. In other preferred embodiments, apolypeptide is dystropin, a mutant FGF receptor, a mutant PDGF receptor,a mutant I-CAM or a mutant V-CAM.

Delivering is preferably injecting an adenovirus vector constructdirectly into a cardiac or a vascular smooth muscle cell. In aparticularly preferred embodiment, delivering is infusing an adenovirusvector construct into a blood vessel that perfuses a muscle cell.

An adenovirus vector construct is typically delivered as apharmaceutical composition. Such a composition comprises aphysiologically acceptable carrier and an effective expression-inducingamount of an adenovirus vector construct.

In yet another aspect, the present invention provides a process forpreparing an adenovirus vector construct for use in transfecting acardiac muscle cell or a vascular smooth muscle cell, which processcomprises the steps of:

a) providing an adenovirus; and

b) incorporating into that adenovirus a coding sequence that encodes agene product desired for introduction into a cardiac muscle or vascularsmooth muscle cell.

In yet another aspect, the present invention provides for an adenovirusvector construct produced in accordance with such a process.

DETAILED DESCRIPTION OF THE INVENTION

I. The Invention

The present invention addresses one or more shortcomings in the priorart through the provision of an adenovirus mediated process forregulating gene product expression in cardiac or vascular smooth musclecells. In accordance with a process of this invention, an adenovirusvector construct is used to deliver a gene to those muscle cells andthus affect expression of that gene 's product. Expression of the geneproduct thereby alters function of those cells and achieves aphysiologically desirable effect. A process of the present invention canbe used to regulate expression of a cardiac muscle cell or a vascularsmooth muscle cell, whether that cell is situated in vitro, in situ, orin vivo in a living organism.

A. Process of Regulating Gene Product Expression in Cardiac Muscle Cell

In one aspect, the present invention provides a process of regulatinggene product expression in a muscle cell of heart (i.e., a cardiacmuscle cell or cardiac myocyte). In accordance with that process, anadenovirus vector construct comprising a coding sequence that encodesthe gene product is delivered to a cardiac muscle cell. The cardiacmuscle cell is then maintained under physiological conditions and for aperiod of time sufficient for the gene to enter the cardiac muscle cell,for the gene to be transcribed and for the product of that gene to beexpressed.

1. Adenovirus Vector

The use of adenovirus as a vector for cell transfection is well known inthe art. Adenovirus vector-mediated cell transfection has been reportedfor various cells (Stratford-Perricaudet, et al., 1992).

An adenovirus vector of the present invention is replication defective.A virus is rendered replication defective by deletion of the viral earlygene region 1 (E1). An adenovirus lacking an E1 region is competent toreplicate only in cells, such as human 293 cells, which expressadenovirus early gene region 1 genes from their cellular genome. Thus,such an adenovirus cannot kill cells that do not express that early geneproduct.

In a preferred embodiment, an adenovirus vector used in the presentinvention is lacking both the E1 and the E3 early gene regions.Techniques for preparing replication defective adenoviruses are wellknown in the art (See. e.g. McGrory et al., 1988, and Gluzman et al.,1982).

It is believed that any adenovirus vector can be used in the practice ofthe present invention. Thus, an adenovirus vector can be of any of the42 different known serotypes or subgroups A-F. Adenovirus type S ofsubgroup C is the preferred staring material for production of areplication-defective adenovirus vector.

An adenovirus is engineered to contain a coding DNA sequence for use asa vector. Such a recombinant adenovirus has been described by Gluzman etal., 1982. Individual DNA sequences such as cDNAs that encode a geneproduct are inserted into the adenovirus to create a vector construct.

In a preferred embodiment, therefore, a coding sequence for a geneproduct is introduced or incorporated into an adenovirus at the positionfrom which the E1 coding sequences have been removed. However, theposition of insertion within the adenovirus sequences is not critical tothe present invention. A coding sequence can also be inserted in lieu ofthe delected E3 region in E3 replacement vectors as described previouslyby Karlsson et al. (1986). Preferably, the E1 region of adenovirus isreplaced by the coding DNA sequence or gene.

The resulting adenovirus vector is co-transfected into 293 cellstogether with a plasmid carrying a complete adenovirus genome topropagate the adenovirus. An exemplary such plasmid is pJM17.Co-transfection is performed in accordance with standard procedures wellknown in the art. By way of example, 293 cells are cultured inDulbecco's modified Eagle's medium containing fetal calf serum.Confluent cultures are split the day before calcium phosphatecotransfection of plasmids. After addition of the DNA to the cells, thecells are shocked (e.g., a 15% glycerol shock) to boost transfectionefficiency and the cells are overlaid with agar in DMEM containing fetalcalf serum, penicillin, streptomycin sulfate, and other antibiotics orantifungal agents as needed. Monolayers are incubated until virl plaquesappear (about 5-15 days).

These plaques are picked, suspended in medium containing fetal calfserum, and used to infect a new monolayer of 293 cells. When greaterthan 90% of the cells showed infection, viral lysates are subjected to afreeze/thaw cycle and designated as primary stocks. The presence ofrecombinant virus is verified by preparation of viral DNA from infected293 cells, restriction analysis, and Southern blotting. Secondary stocksare subsequently generated by infecting 293 cells with primary virusstock at a multiplicity of infection of 0.01 and incubation until lysis.

The particular cell line used to propagate the recombinant adenovirusesof the present invention is not critical to the present invention.Recombinant adenovirus vectors can be propagated on, e.g., human 293cells, or in other cell lines that are permissive for conditionalreplication-defective adenovirus infection, e.g., those which expressadenovirus E1 gene products “in trans” so as to complement the defect ina conditional replication-defective vector. Further, the cells can bepropagated either on plastic dishes or in suspension culture, in orderto obtain virus stocks thereof.

2. Coding Sequence

A coding sequence in an adenovirus vector can code for any gene product.In one embodiment, a gene product is a polynucleodde such as a DNA or aRNA molecule. In an especially preferred embodiment, a gene product isan antisense molecule and, more preferably, an antisense RNA molecule.In a particularly preferred embodiment, an antisense RNA is an antisenseRNA to an oncogene and, preferably, the c-myb oncogene.

In another preferred embodiment, a gene product is a polypeptide. Asused herein, the term “polypeptide” means a polymer of amino acidsconnected by amide linkages, wherein the number of amino acid residuescan range from about 5 to about one million. Preferably, a polypeptidehas from about 10 to about 1000 amino acid residues and, even morepreferably from about 20 to about 500 amino residues. Thus, as usedherein, a polypeptide includes what is often referred to in the art asan oligopeptide (5-10 amino acid residues), a polypeptide (11-100 aminoacid residues) and a protein (>100 amino acid residues).

A polypeptide encoded by a coding sequence can undergopost-translational modification to form conjugates with carbohydrates,lipids, nucleic acids and the like to form glycopolypeptides (e.g.glycoproteins), lipopolypeptides (e.g. lipoproteins) and other likeconjugates.

Any polypeptide can be encoded by a coding sequence of an adenovirus ofthe present invention. A coding sequence can comprise introns and exonsso long as the coding sequence comprises at least one open reading framefor transcription, translation and expression of that polypeptide. Thus,a coding sequence can comprise a gene, a split gene or a cDNA molecule.In the event that the coding sequence comprises a split gene (containsone or more introns), a cell transformed or transfected with a DNAmolecule containing that split gene must have means for removing thoseintrons and splicing together the exons in the RNA transcript from thatDNA molecule if expression of that gene product is desired.

In a preferred embodiment, a polypeptide encoded by a coding sequence ofan adenovirus vector construct of the present invention alters thefunction of a cardiac or vascular smooth muscle cell exposed to thatpolypeptide. Exemplary and preferred polypeptides are growth factorssuch as an endothelial growth factor or a vascular smooth muscle growthfactor; a growth factor receptor; dystropin or a cell adhesion molecule(CAM) such as a mutant I-CAM or V-CAM (Edelman, GM, 1983).

A preferred growth factor is a fibroblast growth factor (FGF) such asFGF-5, acidic FGF and basic FGF; or platelet derived growth factor(PDGF).

A preferred receptor is mutant PDGF receptor (See e.g., Fantl et al.,1989; Coughlin et al., 1990 and Escobedo et al., 1988) or mutant FGFreceptor (See e.g., Peters et al., 1992; Mohammadi et al., 1992; Amayaet al., 1991 and Bellot et al., 1991).

3. Enhancer-Promoter

A coding sequence of an adenovirus vector construct is preferablyoperatively linked to an enhancer-promoter other than an adenovirusenhancer-promoter. A promoter is a region of a DNA molecule typicallywithin about 100 nucleotide pairs in front of (upstream of) the point atwhich transcription begins (i.e., a transcription start site). Thatregion typically contains several types of DNA sequence elements thatare located in similar relative positions in different genes. As usedherein, the term “promoter” includes what is referred to in the art asan upstream promoter region, a promoter region or a promoter of ageneralized eukaryotic RNA Polymerase II transcription unit.

Another type of discrete transcription regulatory sequence element is anenhancer. An enhancer provides specificity of time, location andexpression level for a particular encoding region (e.g., gene). A majorfunction of an enhancer is to increase the level of transcription of acoding sequence in a cell that contains one or more transcriptionfactors that bind to that enhancer. Unlike a promoter, an enhancer canfunction when located at variable distances from transcription startsites so long as a promoter is present.

As used herein, the phrase “enhancer-promoter” means a composite unitthat contains both enhancer and promoter elements. An enhancer-promoteris operatively linked to a coding sequence that encodes at least onegene product. As used herein, the phrase “operatively linted” means thatan enhancer-promoter is connected to a coding sequence in such a waythat the transcription of that coding sequence is controlled andregulated by that enhancer-promoter. Means for operatively linking anenhancer-promoter to a coding sequence are well known in the art. As isalso well known in the art, the precise orientation and locationrelative to a coding sequence whose transcription is controlled, isdependent inter alia upon the specific nature of the enhancer-promoter.Thus, a TATA box minimal promoter is typically located from about 25 toabout 30 base pairs upstream of a transcription initiation site and anupstream promoter element is typically located from about 100 to about200 base pairs upstream of a transcription initiation site. In contrast,an enhancer can be located downstream from the initiation site and canbe at a considerable distance from that site.

An enhancer-promoter used in a vector construct of the present inventioncan be any enhancer-promoter that drives expression in a cardiac orvascular smooth muscle cell. In an example set forth hereinafter, thehuman cytomegalovirus (CMV) immediate early gene promoter has been usedto result in high-level expression of a gene. However, the use of otherviral or mammalian cellular promoters which are well-known in the art isalso suitable to achieve expression of the gene product provided thatthe levels of expression are sufficient to achieve a physiologic effect.Exemplary and preferred enhancer-promoters are the CMV promoter, theRous sarcoma virus (RSV) promoter and the muscle-specific creatinekinase (MCK) enhancer (Zambetti et al., 1992; Yi et al., 1991 andSternberg et al., 1988).

By employing an enhancer-promoter with well-known properties, the leveland pattern of gene product expression can be optimized. For example,selection of an enhancer-promoter that is active specifically in cardiaccells permits tissue-specific expression of the gene product. Apreferred cardiac muscle specific enhancer-promoter is a cardiac isoformtroponin C (cTNC) promoter (See e.g., Parmacek et al., 1992 and Parmaceket al., 1990). Still further, selection of an enhancer-promoter that isregulated in response to a specific physiologic signal can permitinducible gene product expression.

4. Transcription-terminating Region

A coding sequence of an adenovirus vector construct is operativelylinked to a transcription terminating region. RNA polymerase transcribesan encoding DNA sequence through a site where polyadenylation occurs.Typically, DNA sequences located a few hundred base pairs downstream ofthe polyadenylation site serve to terminate transcription. Those DNAsequences are referred to herein as transcription-termination regions.Those regions are required for efficient polyadenylation of transcribedmessenger RNA (mRNA).

Transcription-terminating regions are well known in the art. A preferredtranscription-terminating region used in an adenovirus vector constructof the present invention comprises a polyadenylation signal of SV40 orthe protamine gene.

5. Delivering

Means by which an adenovirus vector construct is delivered to a cardiacmuscle cell depend upon whether that cardiac muscle cell is situated invitro, in situ or in vivo. Where a cardiac muscle cell is in vitro,delivery of an adenovirus construct is preferably accomplished by (1)exposing that cell to medium that contains the adenovirus or (2)injecting the adenovirus vector construct into the muscle cell. In apreferred embodiment, delivering is injecting an adenovirus vectorconstruct into a cardiac muscle cell. Where a cardiac muscle cell issituated in situ (e.g. in an intact heart), delivery is preferablyaccomplished by (1) perfusing the in situ preparation with a solutionthat contains an adenovirus vector construct or (2) injecting such aconstruct into a muscle (e.g. ventricle wall, atrial wall). Injection ofa recombinant DNA molecule into the myocardium resulted in expression ofthe gene product encoded by that molecule (U.S. patent application Ser.No. 07/789,983).

As set forth in that patent application, a recombinant bacterial,galactosidase gene under the control of a Rous sarcoma virus promoterwas introduced into and expressed in adult rat cardiac myocytes in vivoby the injection of purified plasmid DNA directly into the leftventricular wall. Cardiac myocytes expressing the recombinantβ-galactosidase activity were detected histochemically in rat heart forat least six months following injection of the recombinantβ-galactosidase gene.

Only cardiac muscle cells were found to have taken up the vector andexpressed β-gal. No expression of β-gal was observed in other cells inthe heart such as fibroblasts or the cells lining the heart bloodvessels. More than about 75 percent of the hearts receiving the injectedDNA expressed the foreign gene, and this expression was stable forperiods of at least six months.

By way of further example, the direct injection of a gene for fibroblastgrowth factor-5 (an angiogenesis factor gene) was associated with anincrease in capillary number in the injected heart. Following injectionof a recombinant plasmid comprising FGF-5 DNA, a 30-40 percent increasein the number of capillaries was observed in the injected heart wall ascompared to hearts injected with control DNA solutions. Microscopicexamination revealed that the structure of the capillaries in theinjected hearts was normal.

Rats were injected with a plasmid-encoding human fibroblast growthfactor-5 (hFGF-5) in an attempt to stimulate angiogenesis or collateralblood flow in the adult rat heart. Rats were sacrificed at 3 weeksfollowing injection and capillary density was measured by computerizedlight microscopy. Rats injected with control vectors displayedapproximately 2300 capillaries/mm² (p<0.001). Thus, direct injection ofa fibroblast growth factor-5 expression vector stimulates collateralvessel formation in areas of injected myocardium.

Where a cardiac muscle cell is situated in vivo, delivery is preferablyaccomplished by (1) infusing an adenovirus vector construct into a bloodvessel that perfuses the heart or (2) injecting an adenovirus vectorconstruct directly into a heart muscle such as a ventricular or atrialwall. In an especially preferred in vivo embodiment, a catheter isinserted into a blood vessel in the neck of an organism and the tip ofthe indwelling catheter is advanced with fluoroscopic guidance to aposition in a coronary artery or coronary sinus that perfuses a portionof the myocardium. It is preferred that the tip of an indwellingcatheter be placed in proximity to an area of the heart that containscardiac cells to be transfected. Thus, in one preferred embodiment, anindwelling catheter is inserted into either an external jugular vein ora carotid artery and the catheter is advanced until the tip of thecatheter is situated in the coronary sinus ostium or left coronaryartery. By way of example, where cardiac muscle cells were transfectedwith an adenovirus vector construct comprising a coding sequence forβ-galactosidase (β-gal) by infusing that construct via a catheter placedinto the left coronary artery or coronary sinus ostium, cardiac musclecells expressed β-gal (See Examples 1 and 2 hereinafter).

After delivery of an adenovirus vector construct to a cardiac musclecell, that cell is maintained under physiological conditions and for aperiod of time sufficient for the adenovirus vector construct to infectthe cardiac cell and for cellular expression of a coding sequencecontained in that construct.

Physiological conditions are those necessary for viability of thecardiac muscle cell and include conditions of temperature, pH,osmolality and the like. In a preferred embodiment, temperature is fromabout 20° C. to about 50° C., more preferably from about 30° C. to about40° C. and, even more preferably about 37° C.

pH is preferably from about a value of 6.0 to a value of about 8.0, morepreferably from about a value of about 6.8 to a value of about 7.8 and,most preferably about 7.4. Osmolality is preferably from about 200milliosmols per liter (mosm/L) to about 400 mosm/l and, more preferablyfrom about 290 mosm/L to about 310 mosm/L. Other physiologicalconditions needed to sustain cardiac muscle cell viability are wellknown in the art.

A time period sufficient for expression of a coding sequence in acardiac cell varies inter alia as is well known in the art on the typeof adenovirus vector used and the method of delivery. Where theadenovirus vector was derived from type 5 adenovirus and the virusvector was infused into a coronary artery, expression was observed 3 to5 days after infusion (See Example 2 hereinafter).

It should also be pointed out that because the adenovirus vectoremployed in replication defective, it is not capable of replicating inthe cells that are ultimately infected. Moreover, it has been found thatthe genomic integration frequency of adenovirus is usually fairly low.Thus, where continued treatment is required it may be necessary toreintroduce the virus every 6 months to a year. In these circumstances,it may therefore be necessary to conduct long term therapy, whereexpression levels are monitored at selected intervals.

An adenovirus vector construct is typically delivered in the form of apharmacological composition that comprises a physiologically acceptablecarrier and the adenovirus vector construct. An effectiveexpression-inducing amount of an adenovirus vector construct isdelivered. As used herein, the term “effective expression-inducingamount” means that number of virus vector particles necessary toeffectuate expression of a gene product encoded by a coding sequencecontained in that vector. Means for determining an effectiveexpression-inducing amount of an adenovirus vector construct are wellknown in the art. An effective expression-inducing amount is typicallyfrom about 10⁷ plaque forming units (pfu) to about 10¹⁵ pfu, preferablyfrom about 10⁸ pfu to about 10¹⁴ pfu and, more preferably, from about10⁹ to about 10¹² pfu.

As is well known in the art, a specific dose level for any particularsubject depends upon a variety of factors including the infectivity ofthe adenovirus vector, the age, body weight, general health, sex, diet,time of administration, route of administration, rate of excretion, andthe severity of the particular disease undergoing therapy.

In that adenovirus is a virus that infects humans, there can be certainindividuals that have developed antibodies to certain adenovirusproteins. In these circumstances, it is possible that such individualsmight develop an immunological reaction to the virus. Thus, where animmunological reaction is believed to be a possibility, one can firsttest the subject to determine the existence of antibodies. Such a testcan be performed in a variety of accepted manners, for example, througha simple skin test or through a test of the circulating blood levels ofadenovirus-neutralizing antibodies. In fact, under such circumstances,one may desire to introduce a test dose of on the order of ×10⁶ to 1×10⁶or so virus particles. Then, if no untoward reaction is seen, the doseis elevated over a period of time until the desired dosage is reached,such as through the administration of incremental dosages ofapproximately an order of magnitude.

B. Process of Regulating Gene Product Expression in a Vascular SmoothMuscle Cell

In another aspect, the present invention provides a process ofregulating gene product expression in a vascular smooth muscle cell(e.g. an arterial smooth muscle cell). In accordance with that process,an adenovirus vector construct comprising a coding sequence that encodesthat gene product is delivered to a vascular smooth muscle cell. Thevascular smooth muscle cell is then maintained under physiologicalconditions and for a period of time sufficient for the gene to enter thevascular smooth muscle cell, for the gene to be transcribed and for theproduct of that gene to be expressed. A preferred vascular smooth musclecell is a coronary artery smooth muscle cell.

An adenovirus vector construct used in a process of regulating geneproduct expression in a vascular smooth muscle cell is the same as theconstruct discussed above relative to cardiac muscle. Similarly, anenhancer-promoter, a transcription terminating region and a codingsequence are the same as set forth above with the exception that apreferred enhancer-promoter for use with vascular smooth muscle is avascular smooth muscle specific enhancer-promoter such as an endothelinpromoter (See e.g., Lee, et al., 1990 and Bloch et al., 1989) or asmooth muscle α-actin promoter (See e.g., Foster et al., 1992 and Blanket al., 1992).

Delivering is preferably the same as set forth above in relation tocardiac muscle and depends upon whether the vascular smooth muscle cellis situated in situ, in vitro or in vivo.

C. Pharmaceutical Compositions

In another aspect, the present invention relates to a pharmaceuticalcomposition wherein the adenovirus vector gene construct is dispersed ina physiologically acceptable solution or buffer.

A composition of the present invention is typically administeredparenterally in dosage unit formulations containing standard, well knownnontoxic physiologically acceptable carriers, adjuvants, and vehicles asdesired. The term parenteral as used herein includes intravenous,intramuscular, intraarterial injection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions are formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed arewater, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

Preferred carriers include neutral saline solutions buffered withphosphate, lactate, Tris, and the like. Of course, one purifies thevector sufficiently to render it essentially free of undesirablecontaminant, such as defective interfering adenovirus particles orendotoxins and other pyrogens such that it does not cause any untowardreactions in the individual receiving the vector construct. A preferredmeans of purifying the vector involves the use of buoyant densitygradients, such as cesium chloride gradient centrifugation.

D. Process of Preparing an Adenovirus Vector Construct

In another aspect, the present invention provides a process of preparingan adenovirus vector construct for use in regulating gene productexpression in a cardiac muscle or vascular smooth muscle cell. Such aprocess comprises the steps of:

a) providing an adenovirus; and

b) incorporating into that adenovirus a coding sequence that encodes agene product desired for introduction into a cardiac muscle or vascularsmooth muscle cell.

A coding sequence is incorporated into an adenovirus using standardtechniques well known in the art such that the gene product encoded bythat sequence is expressed in a muscle cell infected with the adenovirusvector construct. In a preferred embodiment, a coding sequence isoperatively linked to a non-adenovirus enhancer-promoter. Exemplary andpreferred such enhancer-promoters are set forth above.

A gene product desired for introduction into a cardiac muscle orvascular smooth muscle cell is preferably the same as set forth above inrelation to a process of regulating gene product expression in thosecells.

In yet another aspect, the present invention provides for an adenovirusvector construct produced in accordance with such a process.

The following examples illustrate particular embodiments of the presentinvention and are not limiting of the specification and claims in anyway.

EXAMPLE 1

Making of Adenovirus Vector Construct

This example describes the use of recombinant replication defectiveadenoviruses in the preparation of virus vector constructs comprising acoding DNA sequence.

Recombinant adenovirus (Gluzman et al., 1982) containing distinct cDNAs(AdCMV-cDNA) were prepared in accordance with standard techniques wellknown in the art. E. coli β-galactosidase cDNA carrying the SV40 Tantigen nuclear targeting signal (Bonnerot et al., 1987) was insertedinto pAdCMV to create a distinct construct comprising thecytomegalovirus (CMV) promoter, the β-Gal cDNA and a polyadenylationsignal from either the SV40 virus or the mouse protamine gene, andflanked by adenovirus type 5 sequences. In this construct, the E1 and E3region of adenovirus were deleted and the E1 region was replaced by theβ-Gal encoding sequence.

The resulting plasmid, designated AdCMVβ-gal, was cotransfected into 293cells. Co-transfection was performed as follows: 293 cells were culturedin Dulbecco's modified Eagle's medium (DMEM) containing 2% fetal calfserum. Confluent dishes were split to non-confluent flasks the daybefore cotransfection with pAdCMVβ-gal. Monolayers were incubated untilthe appearance of viral plaques.

These plaques were picked, suspended in DMEM containing 2% fetal calfserum and used to infect a new monolayer of 293 cells. When greater than90% of the cells showed infection, viral lysates were subjected to afreeze/thaw cycle and were designated as primary stocks. Recombinantvirus with the correct structure was verified by preparation of viralDNA from productively-infected 293 cells, restriction analysis, andSouthern blotting. Secondary stocks were subsequently generated byinfecting 293 cells with primary virus stock and incubation until lysis.

The large scale production of recombinant adenovirus was performed in293 cells. Infected cells were lysed 48 hours post-infection.Virus-containing extracts were centrifuged to remove debris beforeprecipitation of the virus. Virus was collected by centrifugation,resuspended in isotonic medium, purified, and sterilized.

Alternatively, precipitated virus can be resuspended in 50 mM Tris-HClpH 7.8 containing CsCl (d=1.10 g/ml), layered over a step-gradientformed of 2 ml CsCl (d=1.40) and 3 ml of CsCl (d=1.30), and centrifuged2 hours at 20,000 rpm at 10° C. in a Sorvall TH641 rotor. Virus iscollected from the lower interface and dialyzed overnight at 4° C.versus isotonic saline.

EXAMPLE 2

Functional Expression of AdCMVβ-gal

Adult rabbits were anesthetized and a catheter was inserted into theright carotid artery or internal jugular vein. The tip of the catheterwas advanced under fluoroscopic guidance to the left coronary artery orcoronary sinus ostium.

About 1000 μg to 1500 μg of adenovirus vector construct pAdCMVβ-gal,prepared in accordance with the procedures of Example 1, were suspendedin physiologically buffered saline. About 2×10⁹ plaque forming units(pfu) of pAdCMVβ-gal were. infused into the indwelling catheter. Thecatheter was removed, all incisions were closed and the rabbits allowedto recover. Rabbits were killed 5 to about 21 days after injection. Theheart and associated vasculature was removed and examinedhistochemically for β-gal activity as set forth below.

Three-millimeter cross sections of the left ventricle were fixed for 5minutes at room temperature with 1.25% glutaraldehyde in PBS, washedthree times at room temperature in PBS, and stained for β-galactosidaseactivity with X-gal (Biorad) for 4-16 hours as described by Nabel et al.(Nabel, et al., (1989). The 3-mm sections were embedded withglycomethocyrlate, and 4-7 μm sections were cut and counterstained withhematoxylin and eosin as described previously. (Nabel, et al., 1989).Photomicroscopy was performed using Kodak Ektachrome 200 file and LeitzLaborlux D and Wild M8 microscopes. β-Gal activity was observed incoronary vascular smooth muscle and in cardiac muscle cells.

EXAMPLE 3

Regulation of Dystropin Expression In Cardiac Cells of Subjects WithDuchenne's Muscular Dystrophy

A process of the present invention can be used in the treatment of humandisorders via gene therapy, such as, for example, in the treatment ofDuchenne's muscular dystrophy.

Human subjects with Duchenne's muscular dystrophy are initially testedfor the presence of antibodies directed against an adenovirus used toprepare an adenovirus vector construct. If antibodies are present or ifthe patient has a history of allergy to other substances, that patientis preferably given a test dose of from about 10³ to about 10⁶recombinant adenovirus particles.

Recombinant adenovirus comprising a coding sequence for dystropin isprepared and purified by any method that would be acceptable to the Foodand Drug Administration for administration to human subjects and provento have sufficient efficacy and purity for human use.

Adenovirus is administered to patients preferably by means ofintravenous administration in any suitable pharmacological composition,either as a bolus or as an infusion over a period of time. An adenovirusis administered in an effective expression-inducing amount. Typically,it is believed that such an effective amount is from about 10⁸ to about5×10¹² virus particles or pfu.

Patients would remain hospitalized during the trial to monitor acute anddelayed adverse reactions such as an inflammatory reaction.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations can be applied to the composition, processand in the steps or in the sequence of steps of the process describedherein without departing from the concept, spirit and scope of theinvention.

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What is claimed is:
 1. A method for delivering a nucleic acid encoding agene product to a cardiac muscle cell or vascular smooth muscle cellcomprising injecting into myocardium or vascular smooth muscle aneffective expression-inducing amount of a replication-deficientadenovirus vector operatively linked to a coding sequence that encodesthe gene product wherein said gene product is expressed in said cardiacmuscle cell or said vascular smooth muscle cell.
 2. The method of claim1 wherein said gene product is a protein competent to induce collateralblood vessel formation.
 3. The method of claim 2 wherein said geneproduct encodes an angiogenesis factor.
 4. The method of claim 1 whereinsaid vector is injected directly into a ventricle or atrial wall of theheart.
 5. The method of claim 1 wherein about 10⁷ to 10¹⁵ plaque formingunits of said vector are injected into the myocardium or said muscle. 6.The method of claim 5 wherein about 10⁸ to 10¹⁴ plaque forming units ofsaid vector are injected into the myocardium or said muscle.
 7. Themethod of claim 5 wherein about 10⁹ to 10¹² plaque forming units of saidvector are injected into the myocardium or said muscle.
 8. The method ofclaim 1 wherein said replication-deficient adenovirus virus lacks theearly gene region E1.
 9. The method of claim 1 wherein said replicationdeficient adenovirus virus lacks the early gene region E3.
 10. Themethod of claim 1 wherein said gene product encodes a growth factor. 11.The method of claim 10 wherein said growth factor is a fibroblast growthfactor or a platelet derived growth factor.
 12. The method of claim 11wherein said fibroblast growth factor is acidic FGF, basic FGF, orFGF-5.
 13. The method of claim 1 wherein said gene product is anendothelial growth factor or a vascular smooth muscle growth factor. 14.The method of claim 1 wherein said gene product is an antisensemolecule.
 15. The method according to claim 14 wherein said antisensemolecule is a c-myb antisense molecule.
 16. The process according toclaim 1 wherein the coding sequence is operatively linked to cardiacmuscle-specific enhancer promoter.
 17. The method according to claim 16wherein said cardiac muscle-specific enhancer-promoter is a cTNCpromoter.
 18. The method according to claim 1 wherein said codingsequence is encoded by cDNA or genomic DNA.
 19. The method according toclaim 1 wherein said coding sequence is operatively linked to anenhancer-promoter other than an adenovirus enhancer-promoter.
 20. Themethod according to claim 19 wherein said enhancer-promoter is acytomegalovirus promoter, an SV40 early promoter, a RSV promoter or anMCK enhancer.
 21. The method according to claim 19 wherein saidenhancer-promoter is specific for vascular smooth muscle.
 22. The methodaccording to claim 21 wherein said vascular smooth muscle specificenhancer-promoter is an endothelin promoter or a smooth muscle or actinpromoter.
 23. The method according to claim 1 wherein said adenovirus isadenovirus type
 5. 24. The method according to claim 1 wherein saidadenovirus lacks the early gene regions E1 and E3.
 25. The methodaccording to claim 1 wherein said adenovirus is adenovirus type 5 thatlacks the early gene region E1.
 26. The method according to claim 1wherein said gene product is a polynucleotide.
 27. The method accordingto claim 1 wherein said gene product is a polypeptide.
 28. The methodaccording to claim 27 wherein said polypeptide is a growth factorreceptor, a cell adhesion molecule or dystrophin.
 29. The methodaccording to claim 28 wherein said growth factor receptor is a mutantFGF receptor or a mutant PDGF receptor.
 30. The method according toclaim 28 wherein said cell adhesion molecule is a mutant I-CAM or mutantV-CAM.
 31. The method according to claim 1 wherein said vector isreintroduced at a selected interval.
 32. The method according to claim31 wherein said interval is from about 6 months to about one year.
 33. Amethod of transforming a vascular smooth muscle cell with apolynucleotide comprising injecting an adenovirus vector containing thepolynucleotide into vascular smooth muscle to thereby transform vascularsmooth muscle cells.
 34. A method of transforming a cardiac muscle cellwith a polynucleotide comprising injecting an adenovirus vectorcontaining the polynucleotide into myocardium to thereby transformcardiac muscle cells.